GRAMD4 Regulates PEVD-Induced Cell Apoptosis Inhibiting Virus Replication Via The Endoplasmic Reticulum Stress Pathway

Porcine epidemic diarrhea(PED),caused by porcine epidemic diarrhea virus(PEDV),has caused huge losses in the global pig industry.As an acute infectious disease,it is most harmful to lactating piglets.Infected piglets suffer from severe diarrhea,vomiting and eventually death.Therefore,understanding the interaction between the host and PEDV is particularly important for the prevention and control of the virus.Glucosyltransferase Rab-like GTPase activator and myotubularin-containing structural domain 4(GRAMD4)is a pro-apoptotic protein that replaces p53 to induce apoptosis in mitochondrial cells.However,the relationship between GRAMD4 and PEDV has not been reported.The present study aimed to elucidate the role of GRAMD4 in the process of PEDV infection and concluded that:1.the PEDV non-structural protein 6(Nsp6)interacts with GRAMD4.In this study,immunoprecipitation(co-IP)combined with mass spectrometry(IP/MS)was performed by transfection of Flag-Nsp6 to identify host proteins interacting with PEDV Nsp6,with which GRAMD4 was predicted to interact.Immunoprecipitation by co-transfection of EGFP-GRAMD4 and Flag-Nsp6 showed Nsp6 and GRAMD4 interactions.Mutual co-localization between GRAMD4 and Nsp6 was viewed using laser confocal microscopy,and the results showed that Nsp6 was able to co-localize with GRAMD4.2.Nsp6 degrades GRAMD4 via the endoplasmic reticulum stress-apoptosis pathway.The expression of GRAMD4 in cells overexpressing Nsp6 and infected with PEDV was examined by western blot.The results showed that GRAMD4 was degraded after overexpression of Nsp6 and infection with PEDV.The degradation pathway of GRAMD4 was probed by Western blot after intracellular transfection with Flag-Nsp6 treated with autophagy inhibitor 3-MA,lysosome inhibitor CQ,proteasome inhibitor MG132,and caspase inhibitor Z-VAD-FMK,and the results showed that the caspase inhibitor Z-VAD-FMK blocked the degradation of GRAMD4.In addition,overexpression of Nsp6 followed by treatment with endoplasmic reticulum stress inhibitor was followed by Western blot to detect the expression of endoplasmic reticulum stress-related proteins and apoptosis-related proteins,and the results showed that Nsp6 mediated the degradation of GRAMD4 by apoptosis through PERK and IRE1 pathways.3.GRAMD4 inhibited the proliferation of PEDV.GRAMD4 overexpression plasmid was constructed as well as synthetic si GRAMD4 to detect PEDV proliferation in cells by q PCR,Western blot,TCID50 and indirect immunofluorescence assay,and the results showed that GRAMD4 overexpression effectively reduced the m RNA and protein expression of PEDV N,while knockdown of GRAMD4 using si RNA promoted m RNA and protein expression of PEDV N.Compared with the control group,the proliferation ability of PEDV was significantly reduced in the overexpression group and significantly enhanced in the knockdown group.4.GRAMD4 promotes PEDV-induced endoplasmic reticulum stress.marc-145 cells overexpressed GRAMD4 overexpression plasmid,and three pathway-related proteins involved in endoplasmic reticulum stress were detected by western blot.The results showed that overexpression of GRAMD4 increased the levels of GRP78,phosphorylated PERK(p-PERK),and phosphorylated IRE1(p-IRE1),but did not alter the expression level of ATF6.In contrast,GRAMD4 knockdown reduced the level of endoplasmic reticulum stress.5.GRAMD4 promoted PEDV-induced endoplasmic reticulum stress-activated apoptosis.To investigate whether GRAMD4 is involved in endoplasmic reticulum stress-activated apoptosis,Marc-145 cells were overexpressed with GRAMD4 and infected with PEDV,and the changes of GRAMD4 overexpression and related proteins after interference were detected by Western blot.The results showed that GRAMD4 overexpression promoted CHOP and phosphorylated JNK(p-JNK),Bax expression,Caspase9 and Caspase3 cleavage,and inhibited Bcl-2 expression.Knockdown of GRAMD4 had the opposite effect.Finally,the deletion plasmid of GRAM structural domain of GRAMD4 was constructed and cells were transfected to detect the GRAM domain function by Western blot and flow cytometry.The results showed that the deletion of GRAM domain could not cause endoplasmic reticulum stress-mediated apoptosis and inhibit viral replication.In conclusion,these studies reveal a mechanism by which GRAMD4 is associated with ER stress and apoptotic regulation of PEDV replication.Nsp6 acts as a down-regulator of GRAMD4 and facilitates the degradation of GRAMD4.GRAMD4 functions in promoting apoptosis and limiting viral replication,requiring the GRAM structural domain.These findings inform host-PEDV interactions and offer possibilities for PEDV decontamination and prevention.